本发明属于基因工程领域，更具体地说涉及一种慢病毒cag-cmv双启动子改造载体构建及应用。
背景技术：
：增强子,位于结构基因附近，能够增强该基因转录活性的一段dna顺序称为增强子。增强子的作用特点包括：①在转录起始点5'或3'侧均能起作用；②相对于启动子的任一指向均能起作用；③发挥作用与受控基因的远近距离相对无关；④对异源性启动子也能发挥作用；⑤通常具有一些短的重复顺序。正是基于增强子相对于启动子发挥作用无方向性的特点，我们计划构建一种共用增强子的慢病毒载体。本发明所述的双启动子为cag启动子和cmv启动子，他们都有一段共同序列为cmvenhancer.本发明构建的慢病毒cagcmv双启动子改造载体表达框示意如图9：技术实现要素：下面结合附图与具体实施方式对本发明作进一步详细的说明：1.本发明的内容是构建共用增强子反向双启动子慢病毒表达载体；2.本发明采用的技术方案是：一种慢病毒cag-cmv双启动子改造载体构建及应用。所述慢病毒载体plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag由pcr扩增得到minicag-mcs-mcherry-3flag片段替换plkd-cmv-egfp-2a-puro-u6-shrna载体中u6-ccdb得到；所述慢病毒载体plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag由pcr扩增得到egfp-2a-puro-minicmv片段插入载体plenti-cag-mcs-mcherry-3flag中获得。3.本发明还涉及构建所述的慢病毒载体的方法，所述方法包括：(1)片段minicag-mcs-mcherry-3flag的扩增：以质plenti-cag-mcs-mcherry-3flag为模板，设计特异性引物扩增获得。所述扩增片段minicag-mcs-mcherry-3flag的引物序列如下：f:5'-cgcctaatggatccgaattcgtcgaggtgagccccacg-3'r:5'-gggcccgctctagactcgagttatttgtcgtcatcatcctta-3'(2)片段egfp-2a-puro-minicmv的扩增：以质粒plkd-cmv-egfp-2a-puro-u6-shrna为模板，设计特异性引物扩增获得。所述扩增片段egfp-2a-puro-minicmv的引物序列如下：f:5'-cagagatccagtttatcgattcaggcaccgggcttgc-3'r:5'-tgattactattaatacgcgttgatgcggttttggcagtac-3'(3)慢病毒载体plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag的构建：用ecori和xhoi酶切plkd-cmv-g&pr-u6-shrna载体回收大片段和minicag-mcs-mcherry-3flag片段进行无缝克隆得到。(4)慢病毒载体plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag的构建：用clai和mlui酶切载体plenti-cag-mcs-mcherry-3flag得到线性化的质粒和egfp-2a-puro-minicmv片段进行无缝克隆得到。4.本发明的有益效果主要体现在：本发明构建的慢病毒载体具有双启动子—cag和cmv，以绿色荧光蛋白egfp和mcherry作为报告基因，可以用现有的荧光显微镜装置检测，为进一步研究慢病毒载体感染细胞的感染效率提供了实验基础。附图说明附图1:慢病毒载体plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag质粒图谱；附图2:慢病毒载体plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag质粒图谱；附图3:慢病毒载体plkd-cmv-egfp-2a-puro-u6-shrna质粒图谱；附图4:慢病毒载体plenti-cag-mcs-mcherry-3flag质粒图谱；附图5:片段minicag-mcs-mcherry-3flag扩增结果图；m:dnamarker：dl2000，lane1:pcramplificationgofminicag-mcs-mcherry-3flag；附图6:片段egfp-2a-puro-minicmv扩增结果图；m:dnamarker：dl2000，lane1:pcramplificationgofegfp-2a-puro-minicmv；附图7:质粒plkd-cmv-g&pr-u6-shrnaecori/xhoi双酶切凝胶回收结果；m:dnamarker1kb,lane1:digestionofplasmidplkd-cmv-g&pr-u6-shrna；附图8:质粒plenti-cag-mcs-mcherry-3flagclai/mlui双酶切凝胶回收结果；m:dnamarker1kb，lane1:digestionofplasmidplenti-cag-mcs-mcherry-3flag；附图9：本发明构建的慢病毒cagcmv双启动子改造载体表达框示意图。具体实施方式以下涉及的化学试剂和生物制品，如未特别说明都为商业化产品。另外，其他未注明的实验操作按照常规分子生物学操作方法进行。下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：实施例1.构建plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag慢病毒表达质粒1.1minicag-mcs-mcherry-3flag片段扩增：以载体plenti-cag-mcs-mcherry-3flag为模板，以f和r引物(seqidno.1～2)对进行pcr获得2268bp的minicag-mcs-mcherry-3flag片段，切胶回收；pcr体系如下：质粒模板(100ng/μl)2μlprimerstarpolymerase(3.5u/μl)0.5μldntps(10mm；2.5mmeach)4μl2xgcbuffer25μlf(10pmol/μl)1μlr(10pmol/μl)1μlddh2o16.5μlpcr反应条件：1.2使用ecori和xhoi酶切plkd-cmv-g&pr-u6-shrna质粒，2h，37℃：酶切体系如下：2μg(2μl)plkd-cmv-egfp-2a-puro-u6-shrna1μlecori(neb)1μlxhoi(neb)5μl10xcutsmart41μlddh2o50μl合计酶切后得到581bp和7928bp的片段，使用generay胶回收试剂盒(产品目录号：gk2042-500)纯化酶切后的7928bp的质粒大片段；1.3扩增得到的片段minicag-mcs-mcherry-3flag与酶切后的载体大片段无缝克隆成目的载体plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag。体系如下：45ng插入片段(minicag-mcs-mcherry-3flag)79.3ng线性化载体(plkd-cmv-egfp-2a-puro-u6-shrn)1μl无缝克隆酶4μl5x反应bufferupto20μlddh2o混匀后在37℃孵育30分种，然后转移到冰上放置5分钟。将连接后的质粒转化至感受态细胞dh5α中，均匀涂至lb固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。1.4挑取几个单菌落扩大培养并使用generay的无内毒素质粒小量制备试剂盒(目录号：gk2007-20)进行质粒小提。1.5鉴定质粒构建成功。经测序验证基因的插入位点、方向和序列完全正确，测序正确的质粒即为所构建的目的质粒，命名为plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag。2.构建plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag慢病毒表达质粒2.1cmv(无增强子)-egfp-2a-puro片段扩增：以载体plkd-cmv-egfp-2a-puro-u6-shrna为模板，以f1和r1引物(seqidno.3～4)对进行pcr获得1706bp的cmv(无增强子)-egfp-2a-puro片段，切胶回收；pcr体系如下：质粒模板(100ng/μl)2μlprimerstarpolymerase(3.5u/μl)0.5μldntps(10mm；2.5mmeach)4μl2xgcbuffer25μlf(10pmol/μl)1μlr(10pmol/μl)1μlddh2o16.5μlpcr反应条件：2.2使用clai和mlui酶切plenti-cag-mcs-mcherry-3flag质粒，2h，37℃：酶切体系如下：2μg(2μl)plenti-cag-mcs-mcherry-3flag1μlecori(neb)1μlxhoi(neb)5μl10xcutsmart41μlddh2o50μl合计酶切后得到8856bp的片段，使用generay胶回收试剂盒(产品目录号：gk2042-500)纯化酶切后的8856bp的质粒大片段；2.3扩增得到的片段cmv(无增强子)-egfp-2a-puro与酶切后的载体大片段无缝克隆成目的载体plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag。体系如下：34ng插入片段(cmv(无增强子)-egfp-2a-puro)89ng线性化载体(plkd-cmv-egfp-2a-puro-u6-shrn)1μl无缝克隆酶4μl5x反应bufferupto20μlddh2o混匀后在37℃孵育30分种，然后转移到冰上放置5分钟。将连接后的质粒转化至感受态细胞dh5α中，均匀涂至lb固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。2.4挑取几个单菌落扩大培养并使用generay的无内毒素质粒小量制备试剂盒(目录号：gk2007-20)进行质粒小提。2.5鉴定质粒构建成功。经测序验证基因的插入位点、方向和序列完全正确，测序正确的质粒即为所构建的目的质粒，命名为plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag。3.转染效率验证用含10％胎牛血清的高糖dmem培养基于5％co2，37℃恒温培养293t细胞(购于美国atcc细胞库)。取对数期细胞以1×105/孔接种到24孔板培养。待细胞融合度达到70％～80％时更换成opti-mem培养基，1小时后plenti-egfp-2a-puro-cmv-minicag-mcs-mcherry-3flag和plenti-egfp-2a-puro-minicmv-cag-mcs-mcherry-3flag质粒0.8μg经lipo2000试剂，转染到293t细胞中，转染48小时候，荧光显微镜下观察转染效果。序列表<110>和元生物技术(上海)股份有限公司<120>一种慢病毒cag-cmv双启动子改造载体构建及应用<160>8<170>siposequencelisting1.0<210>1<211>38<212>dna<213>人工序列(artificialsynthesis)<400>1<210>2<211>42<212>dna<213>人工序列(artificialsynthesis)<400>2<210>3<211>37<212>dna<213>人工序列(artificialsynthesis)<400>3<210>4<211>40<212>dna<213>人工序列(artificialsynthesis)<400>4<210>5<211>2209<212>dna<213>人工序列(artificialsynthesis)<400>5gtcgaggtgagccccacgttctgcttcactctccc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